Blunt end ligation

Ricardo via methods%40net.bio.net (by nightfallelf from yahoo.com)
Tue Feb 13 09:20:02 EST 2007


Hello, i am having a problem doing a ligation with a
PCR product with the restriction sites (sticky ends)
on the primers. So, as i think the problem might be in
the digestion, i decided to do a blunt end ligation
using the same primers, digesting them and fill it
with klenow. As i never did a blunt end ligation
before i would like to see if you can find any problem
in the protocol i made for this procedure:

(primers for PCR will be phosphorilated to do this)

1. Digest of vector and PCR product with xhoI and ndeI
(both originate sticky ends)
2. Gel extraction of both
3. Make blunt ends with Klenow
4. Dephosphorilate the vector
(And after inactivate the AP 65 degrees for  15 mins)
5. Ligation 1:5 with 5% PEG (final concentration)

This insert size (PCR product) is about 1kb and the
vector about 6kb.

My worst fear here is that, since the insert is big
enough, it might recircularise, even with PEG.. I
already had all kinds of recircularization, even with
one sticky end and one blunt end!!! so i am a lil
traumatized with that. Can you please check if this
procedure is alright and if you have any further
advice to this i would appreciate it.

Ps: I know i could do topo cloning but i would rather
not.
(by the way, I checked the “cleavage close to the end
of DNA fragments” from NEB page so primers should be
alright)


Ricardo


 
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